Retinal microglia exacerbate uveitis by functioning as local antigen-presenting cells.

Autoimmune uveitis is a major cause of blindness in the working-age population of developed countries. Experimental autoimmune uveitis (EAU) depends on activation of interphotoreceptor retinoid-binding protein (IRBP) specific CD4 + effector T cells that migrate systemically and infiltrate into the retina. Following systemic induction of retinal antigen-specific T cells, the development of EAU can be broken down into three phases: early phase when inflammatory cells begin to infiltrate the retina, amplification phase, and peak phase. Although studied extensively, the function of local antigen-presenting cells (APCs) within the retina remains unclear. Two potential types of APCs are present during uveitis, resident microglia and infiltrating CD11c + dendritic cells (DCs). MHC class II (MHC II) is expressed within the retina on both CD11c + DCs and microglia during the amplification phase of EAU. Therefore, we used microglia specific (P2RY12 and TMEM119) and CD11c + DC specific MHC II knockout mice to study the function of APCs within the retina using the conventional and adoptive transfer methods of inducing EAU. Microglia were essential during all phases of EAU development: the early phase when microglia were MHC Il negative, and amplification and peak phases when microglia were MHC II positive. Unexpectedly, retinal infiltrating MHC Il + CD11c + DCs were present within the retina but their antigen-presenting function was not required for all phases of uveitis. Our data indicate microglia are the critical APCs within the retina and an important therapeutic target that can prevent and/or diminish uveitis even in the presence of circulating IRBP-specific CD4 + effector T cells.

Retinal Injury Activates Complement Expression in Müller Cells Leading to Neuroinflammation and Photoreceptor Cell Death.

Retinal detachment (RD) is a neurodegenerative blinding disease caused by plethora of clinical conditions. RD is characterized by the physical separation of retina from the underlying retinal pigment epithelium (RPE), eventually leading to photoreceptor cell death, inflammation, and vision loss. Albeit the activation of complement plays a critical role in the pathogenesis of RD, the retinal cellular source for complement production remains elusive. Here, using C3 tdTomato reporter mice we show that retinal injury upregulates C3 expression, specifically in Müller cells. Activation of the complement cascade results in the generation of proinflammatory cleaved products, C3a and C5a, that bind C3aR and C5aR1, respectively. Our flow cytometry data show that retinal injury significantly upregulated C3aR and C5aR1 in microglia and resulted in the infiltration of peripheral immune cells. Loss of C3, C5, C3aR or C5aR1 reduced photoreceptor cell death and infiltration of microglia and peripheral immune cells into the sub-retinal space. These results indicate that C3/C3aR and C5/C5aR1 play a crucial role in eliciting photoreceptor degeneration and inflammatory responses in RD.

Two point-of-care test-based approaches for the exclusion of deep vein thrombosis in general practice: a cost-effectiveness analysis.

BACKGROUND: In the diagnostic work-up of deep vein thrombosis (DVT), the use of point-of-care-test (POCT) D-dimer assays is emerging as a promising patient-friendly alternative to regular D-dimer assays, but their cost-effectiveness is unknown. We compared the cost-effectiveness of two POCT-based approaches to the most common, laboratory-based, situation. METHODS: A patient-level simulation model was developed to simulate the diagnostic trajectory of patients presenting with symptoms of DVT at the general practitioner (GP). Three strategies were defined for further diagnostic work-up: one based on current guidelines ('regular strategy') and two alternative approaches where a POCT for D-dimer is implemented at the 1) phlebotomy service ('DVT care pathway') and 2) GP practice ('fast-POCT strategy'). Probabilities, costs and health outcomes were obtained from the literature. Costs and effects were determined from a societal perspective over a time horizon of 6 months. Uncertainty in model outcomes was assessed with a one-way sensitivity analysis. RESULTS: The Quality-Adjusted Life Years (QALYs) scores for the three DVT diagnostic work-up strategies were all around 0.43 across a 6 month-time horizon. Cost-savings of the two POCT-based strategies compared to the regular strategy were €103/patient for the DVT care pathway (95% CI: -€117-89), and €87/patient for the fast-POCT strategy (95% CI: -€113-67). CONCLUSIONS: Point-of-care-based approaches result in similar health outcomes compared with regular strategy. Given their expected cost-savings and patient-friendly nature, we recommend implementing a D-dimer POCT device in the diagnostic DVT work-up.

Microglia refine developing retinal astrocytic and vascular networks through the complement C3/C3aR axis.

Microglia, a resident immune cell of the central nervous system (CNS), play a pivotal role in facilitating neurovascular development through mechanisms that are not fully understood. Previous reports indicate a role for microglia in regulating astrocyte density. This current work resolves the mechanism through which microglia facilitate astrocyte spatial patterning and superficial vascular bed formation in the neuroretina during development. Ablation of microglia increased astrocyte density and altered spatial patterning. Mechanistically, we show that microglia regulate the formation of the spatially organized astrocyte template required for subsequent vascular growth, through the complement C3/C3aR axis during neuroretinal development. Lack of C3 or C3aR hindered the developmental phagocytic removal of astrocyte bodies and resulted in increased astrocyte density. In addition, increased astrocyte density was associated with elevated proangiogenic extracellular matrix gene expression in C3- and C3aR-deficient retinas, resulting in increased vascular density. These data demonstrate that microglia regulate developmental astrocyte and vascular network spatial patterning in the neuroretina via the complement axis.

CD47 Deficiency Ameliorates Ocular Autoimmune Inflammation.

Autoimmune uveitis is a sight-threatening ocular inflammatory condition in which the retina and uveal tissues become a target of autoreactive immune cells. The CD47 is a ubiquitously expressed transmembrane protein which plays multiple roles in fundamental cellular functions including phagocytosis, proliferation, and adhesion. Signal regulatory protein alpha (SIRPα), one of the CD47 ligands, is predominantly expressed in myeloid lineage cells such as dendritic cells (DCs) or macrophages, and CD47-SIRPα signaling pathway is implicated in the development of autoimmune diseases. Our current study demonstrates how CD47 depletion is effective in the prevention of experimental autoimmune uveitis (EAU), an animal model of human autoimmune uveitis, in animals deficient of CD47 (CD47-/- ). Systemic suppression of SIRPα+ DCs in animals deficient in CD47 resulted in the inability of autoreactive CD4+ T cells to develop, which is crucial to induction of EAU. Of interest, retinal microglia, the resident immune cell of the retina, express SIRPα, however these cells were not operative in EAU suppression in response to CD47 depletion. These results identify CD47 as a significant regulator in the development of SIRPα+ DCs that is vital to disease induction in EAU.

Efficacy of one-stage cartilage repair using allogeneic mesenchymal stromal cells and autologous chondron transplantation (IMPACT) compared to nonsurgical treatment for focal articular cartilage lesions of the knee: study protocol for a crossover randomized controlled trial.

BACKGROUND: Articular cartilage defects in the knee have poor intrinsic healing capacity and may lead to functional disability and osteoarthritis (OA). "Instant MSC Product accompanying Autologous Chondron Transplantation" (IMPACT) combines rapidly isolated recycled autologous chondrons with allogeneic MSCs in a one-stage surgery. IMPACT was successfully executed in a first-in-man investigator-driven phase I/II clinical trial in 35 patients. The purpose of this study is to compare the efficacy of IMPACT to nonsurgical treatment for the treatment of large (2-8 cm2) articular cartilage defects in the knee. METHODS: Sixty patients will be randomized to receive nonsurgical care or IMPACT. After 9 months of nonsurgical care, patients in the control group are allowed to receive IMPACT surgery. The Knee Injury and Osteoarthritis Outcome Score (KOOS), pain (numeric rating scale, NRS), and EuroQol five dimensions five levels (EQ5D-5 L) will be used to compare outcomes at baseline and 3, 6, 9, 12, and 18 months after inclusion. Cartilage formation will be assessed at baseline, and 6 and 18 months after inclusion using MRI. An independent rheumatologist will monitor the onset of a potential inflammatory response. (Severe) adverse events will be recorded. Lastly, the difference between IMPACT and nonsurgical care in terms of societal costs will be assessed by monitoring healthcare resource use and productivity losses during the study period. A health economic model will be developed to estimate the incremental cost-effectiveness ratio of IMPACT vs. nonsurgical treatment in terms of costs per quality adjusted life year over a 5-year time horizon. DISCUSSION: This study is designed to evaluate the efficacy of IMPACT compared to nonsurgical care. Additionally, safety of IMPACT will be assessed in 30 to 60 patients. Lastly, this study will evaluate the cost-effectiveness of IMPACT compared to nonsurgical care. TRIAL REGISTRATION: NL67161.000.18 [Registry ID: CCMO] 2018#003470#27 [EU-CTR; registered on 26 March 2019] NCT04236739 [ ClinicalTrials.gov ] [registered after start of inclusion; 22 January 2020].

Prolonged intraocular residence and retinal tissue distribution of a fourth-generation compstatin-based C3 inhibitor in non-human primates.

Age-related macular degeneration (AMD) is a leading cause of irreversible vision loss among the elderly population. Genetic studies in susceptible individuals have linked this ocular disease to deregulated complement activity that culminates in increased C3 turnover, retinal inflammation and photoreceptor loss. Therapeutic targeting of C3 has therefore emerged as a promising strategy for broadly intercepting the detrimental proinflammatory consequences of complement activation in the retinal tissue. In this regard, a PEGylated second-generation derivative of the compstatin family of C3-targeted inhibitors is currently in late-stage clinical development as a treatment option for geographic atrophy, an advanced form of AMD which lacks approved therapy. While efficacy has been strongly suggested in phase 2 clinical trials, crucial aspects still remain to be defined with regard to the ocular bioavailability, tissue distribution and residence, and dosing frequency of such inhibitors in AMD patients. Here we report the intraocular distribution and pharmacokinetic profile of the fourth-generation compstatin analog, Cp40-KKK in cynomolgus monkeys following a single intravitreal injection. Using a sensitive surface plasmon resonance (SPR)-based competition assay and ELISA, we have quantified both the amount of inhibitor and the concentration of C3 retained in the vitreous of Cp40-KKK-injected animals. Cp40-KKK displays prolonged intraocular residence, being detected at C3-saturating levels for over 3 months after a single intravitreal injection. Moreover, we have probed the distribution of Cp40-KKK within the ocular tissue by means of immunohistochemistry and highly specific anti-Cp40-KKK antibodies. Both C3 and Cp40-KKK were detected in the retinal tissue of inhibitor-injected animals, with prominent co-localization in the choroid one-month post intravitreal injection. These results attest to the high retinal tissue penetrance and target-driven distribution of Cp40-KKK. Given its subnanomolar binding affinity and prolonged ocular residence, Cp40-KKK constitutes a promising drug candidate for ocular pathologies underpinned by deregulated C3 activation.

Correction: Dendritic cells mediate the anti-inflammatory action of omega-3 long-chain polyunsaturated fatty acids in experimental autoimmune uveitis.

[This corrects the article DOI: 10.1371/journal.pone.0219405.].

Dendritic cells mediate the anti-inflammatory action of omega-3 long-chain polyunsaturated fatty acids in experimental autoimmune uveitis.

We previously showed that dietary omega (ω)-3 long-chain polyunsaturated fatty acids (LCPUFAs) suppress inflammation in mice with experimental autoimmune uveitis (EAU). We have now investigated the role of antigen presenting cells (APCs) in this action of ω-3 LCPUFAs. C57BL/6 mice were fed a diet supplemented with ω-3 or ω-6 LCPUFAs for 2 weeks, after which splenocytes were isolated from the mice and cocultured with CD4+ T cells isolated from mice with EAU induced by injection of a human interphotoreceptor retinoid-binding protein peptide together with complete Freund's adjuvant. The proliferation of and production of interferon-γ and interleukin-17 by T cells from EAU mice in vitro were attenuated in the presence of splenocytes from ω-3 LCPUFA-fed mice as compared with those from mice fed ω-6 LCPUFAs. Splenocyte fractionation by magnetic-activated cell sorting revealed that, among APCs, dendritic cells (DCs) were the target of ω-3 LCPUFAs. Adoptive transfer of DCs from mice fed ω-3 LCPUFAs attenuated disease progression in EAU mice as well as the production of pro-inflammatory cytokines by T cells isolated from these latter animals. The proliferation of T cells from control Balb/c mice was also attenuated in the presence of DCs from ω-3 LCPUFA-fed mice as compared with those from ω-6 LCPUFA-fed mice. Furthermore, T cell proliferation in such a mixed lymphocyte reaction was inhibited by prior exposure of DCs from mice fed an ω-6 LCPUFA diet to ω-3 LCPUFAs in vitro. Our results thus suggest that DCs mediate the anti-inflammatory action of dietary ω-3 LCPUFAs in EAU.

The Challenges and Promise of Complement Therapeutics for Ocular Diseases.

Ocular inflammation is a defining feature of sight threating diseases and its dysregulation can catalyze and or propagate ocular neurodegenerative maladies such as age-related macular degeneration (AMD). The complement system, an intrinsic component of the innate immunity, has an integral role in maintaining immune-surveillance and homeostasis in the ocular microenvironment; however, overstimulation can drive ocular inflammatory diseases. The mechanism for complement disease propagation in AMD is not fully understood, although there is accumulating evidence showing that targeted modulation of complement-specific proteins has the potential to become a viable therapeutic approach. To date, a major focus of complement therapeutics has been on targeting the alternative complement system in AMD. Recent studies have outlined potential complement cascade inhibitors that might mitigate AMD disease progression. First-in-class complement inhibitors target the modulation of complement proteins C3, C5, factor B, factor D, and properdin. Herein, we will summarize ocular inflammation in the context of AMD disease progression, current clinical outcomes and complications of complement-mediated therapeutics. Given the need for additional therapeutic approaches for ocular inflammatory diseases, targeted complement modulation has emerged as a leading candidate for eliminating inflammation-driven ocular maladies.

Retinal microglia initiate neuroinflammation in ocular autoimmunity.

Autoimmune uveitis is a sight-threatening ocular inflammatory condition in which the retina and uveal tissues become a target of autoreactive immune cells. While microglia have been studied extensively in autoimmune uveitis, their exact function remains uncertain. The objective of the current study was to determine whether resident microglia are necessary and sufficient to initiate and amplify retinal inflammation in autoimmune uveitis. In this study, we clearly demonstrate that microglia are essential for initiating infiltration of immune cells utilizing a murine model of experimental autoimmune uveoretinitis (EAU) and the recently identified microglia-specific marker P2ry12. Initiating disease is the primary function of microglia in EAU, since eliminating microglia during the later stages of EAU had little effect, indicating that the function of circulating leukocytes is to amplify and sustain destructive inflammation once microglia have triggered disease. In the absence of microglia, uveitis does not develop, since leukocytes cannot gain entry through the blood-retinal barrier, illustrating that microglia play a critical role in regulating infiltration of inflammatory cells into the retina.

Mouse model of ocular hypertension with retinal ganglion cell degeneration.

OBJECTIVES: Ocular hypertension is a primary risk factor for glaucoma and results in retinal ganglion cell (RGC) degeneration. Current animal models of glaucoma lack severe RGC cell death as seen in glaucoma, making assessment of physiological mediators of cell death difficult. We developed a modified mouse model of ocular hypertension whereby long-lasting elevation of intraocular pressure (IOP) is achieved, resulting in significant reproducible damage to RGCs. RESULTS: In this model, microbeads are mixed with hyaluronic acid and injected into the anterior chamber of C57BL/6J mice. The hyaluronic acid allows for a gradual release of microbeads, resulting in sustained blockage of Schlemm's canal. IOP elevation was bimodal during the course of the model's progression. The first peak occurred 1 hours after beads injection, with an IOP value of 44.69 ± 6.00 mmHg, and the second peak occurred 6-12 days post-induction, with an IOP value of 34.91 ± 5.21 mmHg. RGC damage was most severe in the peripheral retina, with a loss of 64.1% compared to that of untreated eyes, while the midperiphery exhibited a 32.4% loss, 4 weeks following disease induction. CONCLUSIONS: These results suggest that sustained IOP elevation causes more RGC damage in the periphery than in the midperiphery of the retina. This model yields significant and reproducible RGC degeneration.

The Alternative Complement System Mediates Cell Death in Retinal Ischemia Reperfusion Injury.

Ischemia reperfusion (IR) injury induces retinal cell death and contributes to visual impairment. Previous studies suggest that the complement cascade plays a key role in IR injury in several systemic diseases. However, the role of the complement pathway in the ischemic retina has not been investigated. The aim of this study is to determine if the alternative complement cascade plays a role in retinal IR injury, and identify which components of the pathway mediate retinal degeneration in response to IR injury. To accomplish this, we utilized the mouse model of retinal IR injury, wherein the intraocular pressure (IOP) is elevated for 45 min, collapsing the retinal blood vessels and inducing retinal ischemia, followed by IOP normalization and subsequent reperfusion. We found that mRNA expression of complement inhibitors complement receptor 1-related gene/protein-y (Crry), Cd55 and Cd59a was down-regulated after IR. Moreover, genetic deletion of complement component 3 (C3-/-) and complement factor b (Fb-/-) decreased IR-induced retinal apoptosis. Because vascular dysfunction is central to IR injury, we also assessed the role of complement in a model of shear stress. In human retinal endothelial cells (HRECs), shear stress up-regulated complement inhibitors Cd46, Cd55, and Cd59, and suppressed complement-mediated cell death, indicating that a lack of vascular flow, commonly observed in IR injury, allows for complement mediated attack of the retinal vasculature. These results suggested that in retinal IR injury, the alternative complement system is activated by suppression of complement inhibitors, leading to vascular dysfunction and neuronal cell death.

Atypical Protein Kinase C: Breaking Down Barriers in Ocular Disease?

This commentary highlights the article by Lin et al that demonstrates the therapeutic potential of small-molecule atypical protein kinase C inhibitors in inflammatory ocular disease.

Microglia inhibit photoreceptor cell death and regulate immune cell infiltration in response to retinal detachment.

Retinal detachment (RD) is a sight-threatening complication common in many highly prevalent retinal disorders. RD rapidly leads to photoreceptor cell death beginning within 12 h following detachment. In patients with sustained RD, progressive visual decline due to photoreceptor cell death is common, leading to significant and permanent loss of vision. Microglia are the resident immune cells of the central nervous system, including the retina, and function in the homeostatic maintenance of the neuro-retinal microenvironment. It is known that microglia become activated and change their morphology in retinal diseases. However, the function of activated microglia in RD is incompletely understood, in part because of the lack of microglia-specific markers. Here, using the newly identified microglia marker P2ry12 and microglial depletion strategies, we demonstrate that retinal microglia are rapidly activated in response to RD and migrate into the injured area within 24 h post-RD, where they closely associate with infiltrating macrophages, a population distinct from microglia. Once in the injured photoreceptor layer, activated microglia can be observed to contain autofluorescence within their cell bodies, suggesting they function to phagocytose injured or dying photoreceptors. Depletion of retinal microglia results in increased disease severity and inhibition of macrophage infiltration, suggesting that microglia are involved in regulating neuroinflammation in the retina. Our work identifies that microglia mediate photoreceptor survival in RD and suggests that this effect may be due to microglial regulation of immune cells and photoreceptor phagocytosis.

Attenuation of choroidal neovascularization by dietary intake of ω-3 long-chain polyunsaturated fatty acids and lutein in mice.

Dietary ω-3 long-chain polyunsaturated fatty acids (LCPUFAs) and lutein each protect against age-related macular degeneration (AMD). We here examined the effects of ω-3 LCPUFAs and lutein supplementation in a mouse model of AMD. Mice were assigned to four groups: (1) a control group fed an ω-3 LCPUFA-free diet, (2) a lutein group fed an ω-3 LCPUFA-free diet with oral administration of lutein, (3) an ω-3 group fed an ω-3 LCPUFA-supplemented diet, and (4) an ω-3 + lutein group fed an ω-3 LCPUFA-supplemented diet with oral administration of lutein. Mice were fed the defined diets beginning 2 weeks before, and received lutein with an oral gavage needle beginning 1 week before, induction of choroidal neovascularization (CNV) by laser photocoagulation. The area of CNV measured in choroidal flat-mount preparations was significantly reduced in mice fed ω-3 LCPUFAs or lutein compared with those in the control group, and it was reduced in an additive manner in those receiving both ω-3 LCPUFAs and lutein. The concentrations of various inflammatory mediators in the retina or choroid were reduced in mice fed ω-3 LCPUFAs or lutein, but no additive effect was apparent. The generation of reactive oxygen species (ROS) in chorioretinal lesions revealed by dihydroethidium staining as well as the expression of NADPH oxidase 4 (Nox4) in the retina revealed by immunohistofluorescence and immunoblot analyses were attenuated by ω-3 LCPUFAs and lutein in a synergistic manner. Our results thus show that dietary intake of ω-3 LCPUFAs and lutein attenuated CNV in an additive manner and in association with suppression of inflammatory mediator production, ROS generation, and Nox4 expression. Dietary supplementation with both ω-3 LCPUFAs and lutein warrants further study as a means to protect against AMD.

The Complement System Is Critical in Maintaining Retinal Integrity during Aging.

The complement system is a key component of innate immunity comprised of soluble components that form a proteolytic cascade leading to the generation of effector molecules involved in cellular clearance. This system is highly activated not only under general inflammatory conditions such as infections, collagen diseases, nephritis, and liver diseases, but also in focal ocular diseases. However, little is known about the role of the complement system in retinal homeostasis during aging. Using young (6-week-old) and adult (6-month-old) mice in wild type (C57BL/6) and complement knockout strains (C1q-/-, Mbl a/c-/-, Fb-/-, C3-/-, and C5-/-), we compared amplitudes of electroretinograms (ERG) and thicknesses of retinal layers in spectral domain optical coherence tomography between young and adult mice. The ERG amplitudes in adult mice were significantly decreased (p < 0.001, p < 0.0001) compared to that of young mice in all complement knockout strains, and there were significant decreases in the inner nuclear layer (INL) thickness in adult mice compared to young mice in all complement knockout strains (p < 0.0001). There were no significant differences in ERG amplitude or thickness of the INL between young and adult control mice. These data suggest that the complement system plays an important role in maintaining normal retinal integrity over time.

Cytochrome P450 monooxygenase lipid metabolites are significant second messengers in the resolution of choroidal neovascularization.

Age-related macular degeneration (AMD) is the most common cause of blindness for individuals age 50 and above in the developed world. Abnormal growth of choroidal blood vessels, or choroidal neovascularization (CNV), is a hallmark of the neovascular (wet) form of advanced AMD and leads to significant vision loss. A growing body of evidence supports a strong link between neovascular disease and inflammation. Metabolites of long-chain polyunsaturated fatty acids derived from the cytochrome P450 (CYP) monooxygenase pathway serve as vital second messengers that regulate a number of hormones and growth factors involved in inflammation and vascular function. Using transgenic mice with altered CYP lipid biosynthetic pathways in a mouse model of laser-induced CNV, we characterized the role of these lipid metabolites in regulating neovascular disease. We discovered that the CYP-derived lipid metabolites epoxydocosapentaenoic acids (EDPs) and epoxyeicosatetraenoic acids (EEQs) are vital in dampening CNV severity. Specifically, overexpression of the monooxygenase CYP2C8 or genetic ablation or inhibition of the soluble epoxide hydrolase (sEH) enzyme led to increased levels of EDP and EEQ with attenuated CNV development. In contrast, when we promoted the degradation of these CYP-derived metabolites by transgenic overexpression of sEH, the protective effect against CNV was lost. We found that these molecules work in part through their ability to regulate the expression of key leukocyte adhesion molecules, on both leukocytes and endothelial cells, thereby mediating leukocyte recruitment. These results suggest that CYP lipid signaling molecules and their regulators are potential therapeutic targets in neovascular diseases.

Revisiting the mouse model of oxygen-induced retinopathy.

Abnormal blood vessel growth in the retina is a hallmark of many retinal diseases, such as retinopathy of prematurity (ROP), proliferative diabetic retinopathy, and the wet form of age-related macular degeneration. In particular, ROP has been an important health concern for physicians since the advent of routine supplemental oxygen therapy for premature neonates more than 70 years ago. Since then, researchers have explored several animal models to better understand ROP and retinal vascular development. Of these models, the mouse model of oxygen-induced retinopathy (OIR) has become the most widely used, and has played a pivotal role in our understanding of retinal angiogenesis and ocular immunology, as well as in the development of groundbreaking therapeutics such as anti-vascular endothelial growth factor injections for wet age-related macular degeneration. Numerous refinements to the model have been made since its inception in the 1950s, and technological advancements have expanded the use of the model across multiple scientific fields. In this review, we explore the historical developments that have led to the mouse OIR model utilized today, essential concepts of OIR, limitations of the model, and a representative selection of key findings from OIR, with particular emphasis on current research progress.

The alternative complement pathway aids in vascular regression during the early stages of a murine model of proliferative retinopathy.

Proliferative retinopathic diseases often progress in 2 phases: initial regression of retinal vasculature (phase 1) followed by subsequent neovascularization (NV) (phase 2). The immune system has been shown to aid in vascular pruning in such retinopathies; however, little is known about the role of the alternative complement pathway in the initial vascular regression phase. Using a mouse model of oxygen-induced retinopathy (OIR), we observed that alternative complement pathway-deficient mice (Fb(-/-)) exhibited a mild decrease in vascular loss at postnatal day (P)8 compared with age- and strain-matched controls (P = 0.035). Laser capture microdissection was used to isolate the retinal blood vessels. Expression of the complement inhibitors Cd55 and Cd59 was significantly decreased in blood vessels isolated from hyperoxic retinas compared with those from normoxic control mice. Vegf expression was measured at P8 and found to be significantly lower in OIR mice than in normoxic control mice (P = 0.0048). Further examination of specific Vegf isoform expression revealed a significant decrease in Vegf120 (P = 0.00032) and Vegf188 (P = 0.0092). In conjunction with the major modulating effects of Vegf during early retinal vascular development, our data suggest a modest involvement of the alternative complement pathway in targeting vessels for regression in the initial vaso-obliteration stage of OIR.